ABSTRACT Title of Document: EVALUATION OF ANTIBIOTIC-RESISTANT BACTERIA IN TERTIARY TREATED WASTEWATER, RECLAIMED WASTEWATER USED FOR SPRAY IRRIGATION, AND RESULTING OCCUPATIONAL EXPOSURES. Rachel Elizabeth Rosenberg Goldstein, Master of Public Health in Environmental Health, 2010 Directed By: Dr. Amy R. Sapkota, Assistant Professor, Maryland Institute for Applied Environmental Health Occupational exposures to vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) in reclaimed wastewater used for spray irrigation were evaluated. In 2009, wastewater was collected from a tertiary- treatment facility, and reclaimed wastewater, nasal and dermal swab samples from an irrigation site. Samples were evaluated for MRSA and VRE using standard methods, PCR, and susceptibility testing. MRSA and VRE were isolated from all wastewater samples except effluent. While wastewater MRSA isolates were multidrug resistant (98%), no MRSA was isolated in irrigation water or swabs. VRE was isolated in one irrigation water sample. Fewer irrigation workers were colonized with S. aureus (31%) compared to controls (46%), but they harbored more multidrug resistant S. aureus. This is the first study to 1) evaluate antibiotic-resistant bacteria (ARB) in U.S. reclaimed wastewater and resulting occupational exposures, and 2) detect MRSA in U.S. wastewater. The findings suggested that tertiary wastewater treatment effectively reduced MRSA and VRE. EVALUATION OF ANTIBIOTIC-RESISTANT BACTERIA IN TERTIARY TREATED WASTEWATER, RECLAIMED WASTEWATER USED FOR SPRAY IRRIGATION, AND RESULTING OCCUPATIONAL EXPOSURES. By Rachel Elizabeth Rosenberg Goldstein Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Public Health in Environmental Health 2010 Advisory Committee: Dr. Amy R. Sapkota, Chair Dr. Betty J. Dabney Dr. Gary Felton Dr. Sam W. Joseph Dr. Amir Sapkota © Copyright by Rachel Elizabeth Rosenberg Goldstein 2010 Dedication For my mother, and my friend, Ellen Witte Rosenberg, who told me that I have the potential to do and be whatever I want. ii Acknowledgements I would like to thank my husband, Paul Goldstein, for his unwavering support and enthusiasm for my work and everything I do. His love and encouragement have been essential to my ability to grow and prosper throughout my graduate studies. My family (Rosenbergs, Wittes, and Goldsteins) has always supported my education and efforts to improve the environment and public health. Through good times and bad, I have always been able to count on them. Thank you all. Dr. Amy R. Sapkota has been my adviser, mentor, and all-around champion. She has encouraged me to challenge myself and take advantage of opportunities as they arise. With Dr. Sapkota’s support I have been given the chance to engage myself fully in science. She has helped me focus on the big picture and stoked my passion for environmental health. I cannot thank her enough for providing me with these opportunities and her guidance. I would like to thank the rest of my thesis committee, Dr. Betty J. Dabney, Dr. Gary Felton, Dr. Sam W. Joseph, and Dr. Amir Sapkota, for their enthusiasm for my project and their assistance in strengthening my study and my thesis. Dr. Shirley Micallef taught me the basics of microbiology lab work, to find excitement and joy in what you do, and to be proud of your work. Her mentoring and friendship have been invaluable. Mr. Ashish George lent his time and hands to this project in the lab. Thank you. Mr. John Martin and Mr. John Denholm, generously provided their time and knowledge to this project. My thesis would not have been possible without their cooperation. iii Table of Contents Dedication...........................................................................................................................ii
 Acknowledgements............................................................................................................iii
 List of Tables.....................................................................................................................vi
 List of Figures...................................................................................................................vii
 Chapter 1: Introduction.......................................................................................................1
 Water Use........................................................................................................................1
 Microbial Contaminants in Wastewater..........................................................................3
 Public Health Risks.........................................................................................................4
 Research Rationale..........................................................................................................5
 Chapter 2: Background.......................................................................................................6
 Introduction.....................................................................................................................6
 Staphylococcus aureus....................................................................................................6
 Genus Description.......................................................................................................6
 Species Description.....................................................................................................7
 Ecological Habitat and Distribution............................................................................8
 Epidemiology and Pathogenicity..............................................................................10
 Methicillin Resistance...............................................................................................11
 Enterococcus spp..........................................................................................................13
 Genus Description.....................................................................................................13
 Ecological habitat and distribution...........................................................................13
 Epidemiology and Pathogenicity..............................................................................14
 Vancomycin Resistance............................................................................................14
 Wastewater Treatment in the United States..................................................................16
 Bacteria in wastewater..................................................................................................18
 Antibiotic-Resistant Bacteria in Wastewater................................................................19
 Aerosolization of Pathogens at Wastewater Treatment Plants.....................................21
 Seasonal Variations.......................................................................................................22
 Reclaimed Wastewater Use in the United States..........................................................23
 Conclusion....................................................................................................................25
 Chapter 3: Short form manuscript: Reductions of Methicillin-resistant Staphylococcus aureus and Vancomycin-resistant Enterococcus spp. at a U.S. Tertiary Wastewater Treatment Plant.................................................................................................................27
 Abstract.........................................................................................................................27
 Introduction...................................................................................................................27
 Methods.........................................................................................................................28
 Results...........................................................................................................................30
 MRSA and MSSA....................................................................................................30
 VRE and VSE...........................................................................................................30
 Antimicrobial Susceptibility.....................................................................................31
 Discussion.....................................................................................................................32
 Conclusions...................................................................................................................35
 iv Chapter 4: Antibiotic-Resistant Bacteria Present in Reclaimed Wastewater and Occupational Exposure at a Spray Irrigation Site.............................................................43
 Abstract.........................................................................................................................43
 Introduction...................................................................................................................44
 Goals.........................................................................................................................45
 Methods.........................................................................................................................45
 Site Description.........................................................................................................45
 Sample Collection.....................................................................................................46
 Questionnaire............................................................................................................48
 Isolation.....................................................................................................................48
 Identification.............................................................................................................51
 Results...........................................................................................................................53
 Water.........................................................................................................................53
 Air.............................................................................................................................53
 Nasal and Dermal Swabs..........................................................................................54
 Discussion.....................................................................................................................55
 Water.........................................................................................................................55
 Air.............................................................................................................................59
 Nasal and Dermal Swabs..........................................................................................59
 Limitations....................................................................................................................61
 General......................................................................................................................61
 Water.........................................................................................................................62
 Nasal and Dermal Swabs..........................................................................................62
 Conclusions...................................................................................................................64
 Chapter 5: Overall Conclusions and Public Health Implications.....................................70
 Summary.......................................................................................................................70
 Public Health Implications............................................................................................72
 Concluding Thoughts....................................................................................................75
 Appendices........................................................................................................................78
 A. Reclaimed Wastewater Spray Irrigation Project Protocol...........................................78
 B. Participant Questionnaire.............................................................................................94
 Bibliography.....................................................................................................................96
 v List of Tables Table 1: MIC distributions for 12 antibiotics observed among methicillin-resistant Staphylococcus aureus collected from different sampling locations at a tertiary wastewater treatment plant 40 Table 2: Percentage of multidrug resistant methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) (fraction of total isolates per season) 41 40 Table 3: MIC distributions observed among methicillin- susceptible Staphylococcus aureus collected from different sampling locations at a tertiary wastewater treatment plant 42 Table 4: Minimum inhibitory concentrations (MICs) of select Staphylococcus aureus and methicillin-resistant coagulase negative Staphylococcus isolates from spray irrigation and office workers’ nasal and dermal swabs 68 Table 5: Demographic characteristics and risk factors of spray irrigation workers and office worker controls 69 vi List of Figures Figure 1: Typical wastewater treatment plant process steps 26 Figure 2: Colony forming units (CFU) per milliliter (CFU/mL) of vancomycin-susceptible Enterococcus spp. found in selected wastewater samples 37 Figure 3: Colony forming units (CFU) per milliliter (CFU/mL) of vancomycin-resistant Enterococcus spp. found in selected wastewater samples 38 Figure 4: Average number of antibiotics that MRSA isolates display resistance to in different treatment steps of a tertiary wastewater treatment facility in the Mid-Atlantic Region (n = total number of isolates in sample type from both collection dates) 39 Figure 5: Spray irrigation site: reclaimed wastewater treatment and distribution process 65 Figure 6: Concentration of Enterococcus spp. and vancomycin- resistant Enterococcus spp. in reclaimed wastewater used for spray irrigation samples 66 Figure 7: Percentage of methicillin-susceptible Staphylococcus aureus nasal swab isolates from spray irrigation worker and office worker controls expressing resistance to common gram-positive antibiotics 67 Figure 8: Number of Treatment Facilities and Population Served per State by Level of Treatment for Year 2004 76 vii